This invention relates to methods of specifically delivering an effector molecule to a tumor cell. In particular this invention relates to chimeric molecules that specifically bind to IL-13 receptors and their use to deliver molecules having a particular activity to tumors overexpressing IL-13 receptors.
In a chimeric molecule, two or more molecules that exist separately in their native state are joined together to form a single molecule having the desired functionality of all of its constituent molecules. Frequently, one of the constituent molecules of a chimeric molecule is a xe2x80x9ctargeting moleculexe2x80x9d. The targeting molecule is a molecule such as a ligand or an antibody that specifically binds to its corresponding target, for example a receptor on a cell surface. Thus, for example, where the targeting molecule is an antibody, the chimeric molecule will specifically bind (target) cells and tissues bearing the epitope to which the antibody is directed.
Another constituent of the chimeric molecule may be an xe2x80x9ceffector moleculexe2x80x9d. The effector molecule refers to a molecule that is to be specifically transported to the target to which the chimeric molecule is specifically directed. The effector molecule typically has a characteristic activity that is desired to be delivered to the target cell. Effector molecules include cytotoxins, labels, radionuclides, ligands, antibodies, drugs, liposomes, and the like.
In particular, where the effector component is a cytotoxin, the chimeric molecule may act as a potent cell-killing agent specifically targeting the cytotoxin to cells bearing a particular target molecule. For example, chimeric fusion proteins which include interleukin 4 (IL-4) or transforming growth factor (TGFxcex1) fused to Pseudomonas exotoxin (PE) or interleukin 2 (IL-2) fused to Diphtheria toxin (DT) have been shown-to specifically target and kill cancer cells (Pastan et al., Ann. Rev. Biochem., 61: 331-354 (1992)).
Generally, it is desirable to increase specificity and affinity and decrease cross-reactivity of chimeric cytotoxins in order to increase their efficacy. To the extent a chimeric molecule preferentially selects and binds to its target (e.g. a tumor cell) and not to a non-target (e.g. a healthy cell), side effects of the chimeric molecule will be minimized. Unfortunately, many targets to which chimeric cytotoxins have been directed (e.g. the IL-2 receptor), while showing elevated expression on tumor cells, are also expressed at significant levels on healthy cells. Thus, chimeric cytotoxins directed to these targets frequently show adverse side-effects as they bind non-target (e.g., healthy) cells that also express the targeted receptor.
The present invention provides methods and compositions for specifically delivering an effector molecule to a tumor cell. In particular, the present invention provides chimeric molecules that specifically target tumor cells with less binding to healthy cells than other analogous chimeric molecules known in the prior art.
The improved specific targeting of this invention is premised, in part, on the discovery that tumor cells, especially carcinomas such as renal cell carcinoma, overexpress IL-13 receptors at extremely high levels. The extremely high level of IL-13 receptor expression on target tumor cells permits the use of lower dosages of chimeric molecule to deliver the same amount of effector molecule to the target cells and also results in reduced binding of non-tumor cells.
In a preferred embodiment, this invention provides for a method for specifically delivering an effector molecule to a tumor cell bearing an IL-13 receptor. The method involves providing a chimeric molecule comprising an effector molecule attached to a targeting molecule that specifically binds to an IL-13 receptor and contacting the tumor with the chimeric molecule resulting in binding of the chimeric molecule to the tumor cell.
The targeting molecule is preferably either a ligand, such as IL-13 or circularly permuted IL-13 (cpIL-13, especially cpIL-13 where the native IL-13 is opened between residues 43 and 44 (Gly and Met respectively) to produce a cpIL-13 having Met44 as the amino terminus and Gly43 as its carboxyl terminus) that specifically binds an IL-13 receptor, or an anti-IL-13 receptor antibody. The targeting molecule may be conjugated to the effector molecule, or where both targeting and effector molecules are polypeptides, the targeting molecule may be joined to the effector molecule through one or more peptide bonds thereby forming a fusion protein. Suitable effector molecules include a cytotoxin, a label, a radionuclide, a drug, a liposome, a ligand, and an antibody. In a particularly preferred embodiment, the effector is a cytotoxin, more specifically a Pseudomonas exotoxin such as PE38QQR or PE4E. Where the Pseudomonas exotoxin is fused to an IL-13 targeting molecule, preferred fusion proteins include, but are not limited to IL-13-PE38QQR, IL-13-PE4E, cpIL-13-PE38QQR, and cpIL-13-PE4E.
In another embodiment, this invention provides a method for impairing the growth of tumor cells, more preferably solid tumor cells, bearing an IL-13 receptor. The method involves contacting the tumor with a chimeric molecule comprising an effector molecule selected from the group consisting of a cytotoxin, a radionuclide, a ligand and an antibody. The effector molecule is attached to a targeting molecule that specifically binds a human IL-13 receptor. The targeting molecule is preferably a ligand (such as IL-13) that binds the IL-13 receptor or an anti-IL-13 receptor antibody. Preferred cytotoxic effector molecules include Pseudomonas exotoxin, Diphtheria toxin, ricin and abrin. Psuedomonas exotoxins, such as PE38QQR and PE4E, are particularly preferred. The targeting molecule may be conjugated or fused to the effector molecule with attachment by fusion preferred for cytotoxic effector molecules. The tumor growth that is impaired may be tumor growth in a human. Thus the method may further comprise administering the chimeric molecule to a human intravenously into a body cavity, or into a human or an organ.
In yet another embodiment, this invention provides for a method of detecting the presence or absence of a tumor. The method involves contacting the tumor with a chimeric molecule comprising a detectable label attached to a targeting molecule that specifically binds a human IL-13 receptor and detecting the presence or absence of the label. In a preferred embodiment, the label is selected from the group consisting of a radioactive label, an enzymatic label, an electron dense label, and a fluorescent label. Preferred targeting molecules include, but are not limited to IL-13, cpIL-13, and anti-IL-13R antibodies.
This invention also provides for vectors comprising a nucleic acid sequence encoding a chimeric polypeptide fusion protein comprising an IL-13, or a cpIL-13, attached to a second polypeptide. The chimeric polypeptide fusion protein specifically binds to a tumor cell bearing an IL-13 receptor. A preferred vector encodes an IL-13-PE or cpIL-13-PE fusion protein and more preferably encodes an IL-13-PE38QQR, IL-13-PE4E, cpIL-13-PE38QQR, or cpIL-13-PE4E fusion protein.
This invention also provides for host cells comprising a nucleic acid sequence encoding a chimeric polypeptide fusion protein comprising an IL-13 attached to a second polypeptide. A preferred host cell comprises a nucleic acid encoding an IL-13-PE, or cpIL-13-PE, fusion protein, more preferably encoding an IL-13-PE38QQR, IL-I3-PE4E, cpIL-13-PE38QQR, or cpIL-13-PE4E fusion protein. The encoded fusion protein specifically binds to a tumor cell bearing an IL-13 receptor. Particularly preferred host cells are bacterial host cells, especially E. coli cells.
In still yet another embodiment, this invention provides chimeric molecules that specifically bind a tumor cell bearing an IL-13 receptor. In one preferred embodiment, the chimeric molecule comprises a cytotoxic molecule attached to a targeting molecule that specifically binds an IL-13. The targeting molecule may be conjugated or fused to the cytotoxic molecule. In a preferred embodiment, the targeting molecule is fused to the cytotoxin thereby forming a single-chain fusion protein. Particularly preferred targeting molecules are IL-13, cpIL-13, or an antibody that specifically binds to the IL-13 receptor. Preferred cytotoxic molecules include Pseudomonas exotoxin, Diphthena toxin, ricin, and abrin, with Pseudomonas exotoxins (especially PE38QQR or PE4E) being most preferred.
In another preferred embodiment, the chimeric molecule comprises an effector molecule attached to an antibody that specifically binds to an IL-13 receptor. Effector molecules include a cytotoxin, a label, a radionuclide, a drug, liposome, a ligand and an antibody. The effector molecule may be fused or conjugated to the antibody.
The invention additionally provides for pharmacological compositions comprising a pharmaceutically acceptable carrier and a chimeric molecule where the chimeric molecule comprises and effector molecule attached to a targeting molecule that specifically binds to an IL-13 receptor. The targeting and effector molecules may be conjugated or fused to each other. Particularly preferred targeting molecules include IL-13, cpIL-13, and anti-IL-13 receptor antibodies, while preferred effector molecules include a cytotoxin, a label, a radionuclide, a drug, a liposome, a ligand and an antibody. A preferred pharmacological composition includes an IL-13-PE fusion protein, more preferably a IL-13-PE38QQR, IL-13-PE4E, cpIL-13-PE38QQR, or cpIL-13-PE4E fusion protein.
Definitions
The term xe2x80x9cspecifically deliverxe2x80x9d as used herein refers to the preferential association of a molecule with a cell or tissue bearing a particular target molecule or marker and not to cells or tissues lacking that target molecule. It is, of course, recognized that a certain degree of non-specific interaction may occur between a molecule and a non-target cell or tissue. Nevertheless, specific delivery, may be distinguished as mediated through specific recognition of the target molecule. Typically specific delivery results in a much stronger association between the delivered molecule and cells bearing the target molecule than between the delivered molecule and cells lacking the target molecule. Specific delivery typically results in greater than 2 fold, preferably greater than 5 fold, more preferably greater than 10 fold and most preferably greater than 100 fold increase in amount of delivered molecule (per unit time) to a cell or tissue bearing the target molecule as compared to a cell or tissue lacking the target molecule or marker.
The term xe2x80x9cresiduexe2x80x9d as used herein refers to an amino acid that is incorporated into a polypeptide. The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass known analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.
A xe2x80x9cfusion proteinxe2x80x9d refers to a polypeptide formed by the joining of two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. The fusion protein may be formed by the chemical coupling of the constituent polypeptides or it may be expressed as a single polypeptide from nucleic acid sequence encoding the single contiguous fusion protein. A single chain fusion protein is a fusion protein having a single contiguous polypeptide backbone.
A xe2x80x9cspacerxe2x80x9d as used herein refers to a peptide that joins the proteins comprising a fusion protein. Generally a spacer has no specific biological activity other than to join the proteins or to preserve some minimum distance or other spatial relationship between them. However, the constituent amino acids of a spacer may be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity of the molecule.
A xe2x80x9cligandxe2x80x9d, as used herein, refers generally to all molecules capable of reacting with or otherwise recognizing or binding to a receptor on a target cell. Specifically, examples of ligands include, but are not limited to, antibodies, lymphokines, cytokines, receptor proteins such as CD4 and CD8, solubilized receptor proteins such as soluble CD4, hormones, growth factors, and the like which specifically bind desired target cells.
The term xe2x80x9ccpIL-13xe2x80x9d is used to designate a circularly permuted (cp) IL-13. Circular permutation is functionally equivalent to taking a straight-chain molecule, fusing the ends (directly or through a linker) to form a circular molecule, and then cutting the circular molecule at a different location to form a new straight chain molecule with different termini.